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1.
Virus Genes ; 55(5): 610-618, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31359359

RESUMO

Current data of hepatitis B virus (HBV) variants associated with treatment outcome identified by next generation sequencing (NGS) are limited. This study was aimed at determining the role of baseline sequence variations in the enhancer II (EnhII), basal core promotor (BCP) and pre-core (PC) regions of HBV genotype C in patients treated with pegylated interferon (PEG-IFN). Patients with HBeAg-positive chronic hepatitis B (CHB) treated with 48-week PEG-IFN were enrolled. Combined response (CR) at week 96 was defined by HBeAg seroconversion plus HBV DNA < 2000 IU/mL and HBsAg < 1000 IU/mL. Pre-treatment viral mutations were characterized by Sanger sequencing and NGS (Miseq Illumina platform). Among 47 patients (32 male, mean age 32.4 years), CR was achieved in 12 (25.5%) individuals. Overall, NGS was superior to Sanger sequencing in detecting mutations (61.7% vs. 38.3%, P < 0.001). Based on NGS, the prevalence of T1753V (T1753C/A/G) and A1762T/G1764A variants were significantly lower in responders compared to non-responders (8.3% vs. 51.4%, P = 0.009 and 33.3% vs. 68.6%, P = 0.032, respectively). No significant difference between groups was found regarding C1653T and G1896A mutants. The absence of T1753V and A1762T/G1764A mutations were factors associated with CR (OR 11.65, 95%CI 1.36-100.16, P = 0.025, and OR 4.36, 95%CI 1.08-17.63, P = 0.039, respectively). The existence of pre-treatment T1753V, A1762T/G1764A mutations and their combination yielded negative predictive values of 94.7%, 85.7% and 93.8%, respectively. The presence of HBV mutants in the BCP region determined by NGS at baseline was associated with poor treatment outcome in patients with HBeAg-positive CHB receiving PEG-IFN.


Assuntos
Antivirais/uso terapêutico , Variação Genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Interferons/uso terapêutico , Mutação , Adolescente , Adulto , Feminino , Genótipo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Regiões Promotoras Genéticas , Resultado do Tratamento , Adulto Jovem
2.
Virologie (Montrouge) ; 23(1): 23-34, 2019 Feb 01.
Artigo em Francês | MEDLINE | ID: mdl-31131827

RESUMO

Hepatitis B virus (HBV) infects approximately 257 million individuals worldwide. Recent advances in the virology and diagnosis of HBV infection are summarized in this review article. A novel classification of the different phases of chronic HBV infection, proposed by the European Association for the Study of the Liver (EASL), is presented. New diagnostic and monitoring tools are now available, including rapid diagnostic tests for hepatitis B surface antigen (HBsAg) detection, HBsAg quantification assays, an HBV core-related antigen (HBcrAg) quantification test, and HBV RNA quantification testing. Their clinical utility under study is discussed in this review.


Assuntos
Técnicas e Procedimentos Diagnósticos/tendências , Vírus da Hepatite B/fisiologia , Hepatite B/diagnóstico , Monitorização Fisiológica/tendências , Virologia/tendências , Biomarcadores/análise , Biomarcadores/sangue , DNA Viral/análise , DNA Viral/sangue , Hepatite B/complicações , Hepatite B/patologia , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/análise , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/análise , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/genética , Antígenos E da Hepatite B/análise , Antígenos E da Hepatite B/sangue , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Humanos , Monitorização Fisiológica/métodos , Virologia/legislação & jurisprudência , Virologia/métodos
3.
Virol J ; 16(1): 43, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30943997

RESUMO

BACKGROUND: Although vaccines for hepatitis B virus (HBV) are highly effective, HBV infections in vaccinees occur. Index samples of breakthrough infections are typically anti-HBc negative but HBV DNA positive with protective anti-HBs levels while HBsAg detection may be delayed or absent. HBsAg mutations have been associated with some vaccine breakthrough cases. METHODS: This research characterizes the serological and molecular profiles of vaccine breakthrough infections in serial samples from two commercially available plasma donor panels. Samples were tested with commercially available assays for HBV antigens and antibodies: HBsAg, HBeAg, anti-HBc, anti-HBc IgM, anti-HBe, and anti-HBs. Different immunoassay approaches for earlier detection of breakthrough infection were explored including hepatitis B core-related antigen (HBcrAg), a research assay for preS2 antigen, and a new prototype ARCHITECT HBsAg assay with improved sensitivity. The prototype HBsAg assay is fully automated and involves no sample pre-treatment. Molecular testing included HBV DNA quantitation and sequencing of preS1, preS2, surface, and basal core promoter/core promoter genes. RESULTS: Although the research preS2 antigen assay allowed earlier detection of the breakthrough infections than current HBsAg assays and HBcrAg, the new prototype ARCHITECT HBsAg assay provided the earliest serologic detection. The ability of the new prototype HBsAg assay to detect HBsAg in the presence of anti-HBs was investigated using known concentrations of native HBsAg mixed with anti-HBs from a vaccinee. The results demonstrated that the prototype ARCHITECT assay is more sensitive in detecting HBsAg in the presence of anti-HBs than current HBsAg assays. Sequencing revealed multiple substitutions in preS1, preS2, and S regions for one panel including a rare D144N substitution associated with vaccine breakthrough that emerged with increasing frequency as the breakthrough infection developed. CONCLUSIONS: When compared with other immunoassay approaches, the new prototype ARCHITECT HBsAg assay allows earlier detection of vaccine breakthrough infections and more sensitive detection of HBsAg in the presence of anti-HBs. Molecular characterization of longitudinal samples demonstrated the progressive appearance of a rare HBsAg mutation associated with vaccine breakthrough.


Assuntos
Doadores de Sangue , Antígenos de Superfície da Hepatite B/genética , Vacinas contra Hepatite B/uso terapêutico , Hepatite B/diagnóstico , Testes Sorológicos , Automação Laboratorial , DNA Viral/análise , Anticorpos Anti-Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Antígenos E da Hepatite B/sangue , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Imunoensaio , Mutação , Sensibilidade e Especificidade
4.
Mol Med Rep ; 19(1): 262-270, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30387827

RESUMO

Hepatitis B virus (HBV) core protein (HBc) serves pivotal roles in the viral life cycle, particularly serving as the basic unit for capsid assembly, and is closely associated with HBV genome replication and progeny virion production. Previous studies have demonstrated that HBc has at least two functional interfaces; two HBc monomers form a homodimer via an intradimer interface, and then 90 or 120 homodimers form an icosahedral capsid via a dimer­dimer interface. In the present study, the role of the HBc dimer­dimer interface in HBV replication was investigated. A panel of residues located at the dimer­dimer interface were identified based on the crystal structure of HBc. Native gel electrophoresis and western blotting revealed that, despite mutations in the dimer­dimer interface, HBc formed a capsid­like structure, whereas mutations at amino acid residues 23­39 completely disrupted capsid assembly. Using denaturing gel electrophoresis, Southern and Northern blotting, and quantitative polymerase chain reaction, it was demonstrated that none of the mutations in the dimer­dimer interface supported pregenomic RNA encapsidation or DNA replication. In addition, these mutants interacted with the wild-type (WT) HBc monomer and inhibited WT genome replication and virion production in a dose­dependent manner. However, the quantity of covalently closed circular DNA in the nucleus was not affected. The present study highlighted the importance of the HBc dimer­dimer interface for normal capsid function and demonstrated that the HBc dimer­dimer interface may be a novel antiviral target.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/química , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Mutação , Multimerização Proteica , Montagem de Vírus , Replicação Viral , Capsídeo , Células Hep G2 , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Humanos , Conformação Proteica
5.
Proc Natl Acad Sci U S A ; 115(48): E11369-E11378, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30420516

RESUMO

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Imunidade Humoral , Falência Hepática Aguda/imunologia , Adulto , Animais , Linfócitos B/imunologia , Feminino , Hepatite B/imunologia , Hepatite B/patologia , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Fígado/imunologia , Fígado/virologia , Falência Hepática Aguda/patologia , Falência Hepática Aguda/virologia , Masculino , Pessoa de Meia-Idade , Pan troglodytes , Linfócitos T/imunologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-30406036

RESUMO

Viral mutations acquired during the course of chronic hepatitis B virus (HBV) infection are known to be associated with the progression and severity of HBV-related liver disease. This study of HBV-infected Saudi Arabian patients aimed to identify amino acid substitutions within the precore/core (preC/C) region of HBV, and investigate their impact on disease progression toward hepatocellular carcinoma (HCC). Patients were categorized according to the severity of their disease, and were divided into the following groups: inactive HBV carriers, active HBV carriers, liver cirrhosis patients, and HCC patients. Two precore mutations, W28* and G29D, and six core mutations, F24Y, E64D, E77Q, A80I/T/V, L116I, and E180A were significantly associated with the development of cirrhosis and HCC. Six of the seven significant core mutations that were identified in this study were located within immuno-active epitopes; E77Q, A80I/T/V, and L116I were located within B-cell epitopes, and F24Y, E64D, and V91S/T were located within T-cell epitopes. Multivariate risk analysis confirmed that the core mutations A80V and L116I were both independent predictors of HBV-associated liver disease progression. In conclusion, our data show that mutations within the preC/C region, particularly within the immuno-active epitopes, may contribute to the severity of liver disease in patients with chronic hepatitis. Furthermore, we have identified several distinct preC/C mutations within the study population that affect the clinical manifestation and progression of HBV-related disease. The specific identity of HBV mutations that are associated with severe disease varies between different ethnic populations, and so the specific preC/C mutations identified here will be useful for predicting clinical outcomes and identifying the HBV-infected patients within the Saudi population that are at high risk of developing HCC.


Assuntos
Carcinoma Hepatocelular/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Mutação de Sentido Incorreto , Adulto , Idoso , Substituição de Aminoácidos , Portador Sadio/virologia , Progressão da Doença , Feminino , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/complicações , Hepatite B Crônica/patologia , Humanos , Cirrose Hepática/complicações , Masculino , Pessoa de Meia-Idade , Arábia Saudita , Adulto Jovem
7.
Sci Rep ; 8(1): 14660, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279478

RESUMO

The HBV core protein self-assembles into particles and encapsidates immune-stimulatory bacterial RNA through a cationic COOH-terminal (C150-183) domain. To investigate if different cationic domains have an impact on the endogenous RNA-binding of HBV-C antigens in mammalian cells, we developed a strep-tag (st) based expression/purification system for HBV-C/RNA antigens in vector-transfected HEK-293 cells. We showed that HBV-stC but not HBV-stC149 particles (lacking the cationic domain) capture low amounts of mammalian RNA. Prevention of specific phosphorylation in cationic domains, either by exchanging the serine residues S155, S162 and S170 with alanines (HBV-stCAAA) or by exchanging the entire cationic domain with a HIV-tat48-57-like sequence (HBV-stC149tat) enhanced the encapsidation of RNA into mutant core particles. Particle-bound mammalian RNA functioned as TLR-7 ligand and induced a Th1-biased humoral immunity in B6 but not in TLR-7-/- mice by exogenous (protein) and endogenous (DNA) vaccines. Compared to core particles, binding of mammalian RNA to freely exposed cationic domains in assembly-deficient antigens was enhanced. However, RNA bound to non-particulate antigens unleash its Th1-stimulating adjuvant activity by DNA- but not protein-based vaccination. Mammalian RNAs targeted by an endogenously expressed antigen thus function as a natural adjuvant in the host that facilitates priming of Th1-biased immune responses by DNA-based immunization.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , RNA/imunologia , Células Th1/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Cátions/metabolismo , DNA Viral/imunologia , Feminino , Vetores Genéticos/genética , Células HEK293 , Hepatite B/imunologia , Hepatite B/prevenção & controle , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vacinas contra Hepatite B/administração & dosagem , Humanos , Imunogenicidade da Vacina , Ligantes , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Domínios Proteicos/genética , Domínios Proteicos/imunologia , RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Th1/metabolismo , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia
8.
Mol Med Rep ; 18(5): 4691-4699, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30221736

RESUMO

Hepatitis B virus (HBV) infection is a global public health problem. T helper (Th)1­associated cytokines are involved in HBV clearance during acute and persistent infection. In our previous study, it was demonstrated that lentiviral vectors encoding ubiquitinated hepatitis B core antigen (LV­Ub­HBcAg) effectively transduced dendritic cells (DCs) to induce maturation, which promoted T cell polarization to Th1 and generated HBcAg­specific cytotoxic T lymphocytes (CTLs) ex vivo. In the present study, HBV transgenic mice were immunized with LV­Ub­HBcAg­transduced DCs and HBcAg­specific immune responses were evaluated. Cytokine expression was analyzed by ELISA. T lymphocyte proliferation was detected with a Cell Counting Kit­8 assay and HBcAg­specific CTL activity was determined using a lactate dehydrogenase release assay. The expression levels of p38­mitogen­activated protein kinase (p38­MAPK), phosphorylated (p)­p38MAPK, c­Jun N­terminal kinase (JNK) and p­JNK were detected by western blot analysis. The results demonstrated that LV­Ub­HBcAg­transduced DCs significantly increased the Th1/Th2 cytokine ratio, and effectively reduced the levels of serum hepatitis B surface antigen (HBsAg), HBV DNA, and liver HBsAg and HBcAg. Furthermore, the LV­Ub­HBcAg­transduced DCs upregulated the expression of p­P38­MAPK and p­JNK in T lymphocytes. In conclusion, the present study indicated that LV­Ub­HBcAg­transduced DCs generated predominant Th1 responses and enhanced CTL activity in HBV transgenic mice. Activation of the P38­MAPK/JNK signaling pathway may be involved in this induction.


Assuntos
Células Dendríticas/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Animais , Diferenciação Celular/imunologia , Células Dendríticas/transplante , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/imunologia , Hepatite B/genética , Hepatite B/terapia , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/administração & dosagem , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Imunização , Lentivirus/genética , Lentivirus/imunologia , MAP Quinase Quinase 4/genética , Camundongos , Camundongos Transgênicos , Transdução de Sinais/imunologia , Células Th1/imunologia , Ubiquitinação , Proteínas Quinases p38 Ativadas por Mitógeno/genética
9.
Antiviral Res ; 159: 1-12, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30201396

RESUMO

Native agarose gel electrophoresis-based particle gel assay has been commonly used for examination of hepatitis B virus (HBV) capsid assembly and pregenomic RNA encapsidation in HBV replicating cells. Interestingly, treatment of cells with several chemotypes of HBV core protein allosteric modulators (CpAMs) induced the assembly of both empty and DNA-containing capsids with faster electrophoresis mobility. In an effort to determine the physical basis of CpAM-induced capsid mobility shift, we found that the surface charge, but not the size, of capsids is the primary determinant of electrophoresis mobility. Specifically, through alanine scanning mutagenesis analysis of twenty-seven charged amino acids in core protein assembly domain and hinge region, we showed that except for K7 and E8, substitution of glutamine acid (E) or aspartic acid (D) on the surface of capsids reduced their mobility, but substitution of lysine (K) or arginine (R) on the surface of capsids increased their mobility in variable degrees. However, alanine substitution of the charged amino acids that are not exposed on the surface of capsid did not apparently alter capsid mobility. Hence, CpAM-induced electrophoresis mobility shift of capsids may reflect the global alteration of capsid structure that changes the exposure and/or ionization of charged amino acid side chains of core protein. Our findings imply that CpAM inhibition of pgRNA encapsidation is possibly due to the assembly of structurally altered nucleocapsids. Practically, capsid electrophoresis mobility shift is a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific CpAMs.


Assuntos
Antivirais/farmacologia , Capsídeo/metabolismo , Vírus da Hepatite B/fisiologia , RNA/metabolismo , Proteínas do Core Viral/genética , Montagem de Vírus , Regulação Alostérica , Proteínas do Capsídeo/metabolismo , Eletroforese , Ensaio de Desvio de Mobilidade Eletroforética , Células Hep G2 , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , RNA Viral/metabolismo , Replicação Viral
10.
J Virol ; 92(20)2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30089690

RESUMO

The hepatitis B virus (HBV) capsid or core protein (Cp) can self-assemble to form an icosahedral capsid. It is now being pursued as a target for small-molecule antivirals that enhance the rate and extent of its assembly to yield empty and/or aberrant capsids. These small molecules are thus called core protein allosteric modulators (CpAMs). We sought to understand the physical basis of CpAM-resistant mutants and how CpAMs might overcome them. We examined the effects of two closely related CpAMs, HAP12 and HAP13, which differ by a single atom but have drastically different antiviral activities, on the assembly of wild-type Cp and three T109 mutants (T109M, T109I, and T109S) that display a range of resistances. The T109 side chain forms part of the mouth of the CpAM binding pocket. A T109 mutant that has substantial resistance even to a highly active CpAM strongly promotes normal assembly. Conversely, a mutant that weakens assembly is more susceptible to CpAMs. In crystal and cryo-electron microscopy (cryo-EM) structures of T=4 capsids with bound CpAMs, the CpAMs preferentially fit into two of four quasi-equivalent sites. In these static representations of capsid structures, T109 does not interact with the neighboring subunit. However, all-atom molecular dynamics simulations of an intact capsid show that T109 of one of the four classes of CpAM site has a hydrophobic contact with the neighboring subunit at least 40% of the time, providing a physical explanation for the mutation's ability to affect capsid stability, assembly, and sensitivity to CpAMs.IMPORTANCE The HBV core protein and its assembly into capsids have become important targets for development of core protein allosteric modulators (CpAMs) as antivirals. Naturally occurring T109 mutants have been shown to be resistant to some of these CpAMs. We found that mutation of T109 led to changes in capsid stability and recapitulated resistance to a weak CpAM, but much less so than to a strong CpAM. Examination of HBV capsid structures, determined by cryo-EM and crystallography, could not explain how T109 mutations change capsid stability and resistance. However, by mining data from a microsecond-long all-atom molecular dynamics simulation, we found that the capsid was extraordinarily flexible and that T109 can impede entry to the CpAM binding site. In short, HBV capsids are incredibly dynamic and molecular mobility must be considered in discussions of antiviral mechanisms.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Mutação , Montagem de Vírus/efeitos dos fármacos , Microscopia Crioeletrônica , Cristalografia por Raios X , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica
11.
J Med Virol ; 90(12): 1875-1881, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30085356

RESUMO

AIM: The worldwide prevalence of hepatitis C virus infection (HCV) is nearly 150 to 170 million cases. The prevalence of HCV infection in India is estimated to be around 1%. In India HCV genotype (GT)3 is the predominant GT followed by GT1. Our study aims to establish the prevalent GTs/subtypes of HCV circulating in Uttar Pradesh, North India, as reported from a tertiary care hospital. METHODS: The study was a retrospective observational analysis of consecutive 404 HCV RNA positive cases referred to our hospital from September 2014 to April 2017, and was approved by an institutional ethics committee. Written informed consent was taken from each participant. Clinical and demographic details of these patients were recorded using predesigned questionnaires. All the laboratory testing was carried out on a stored serum sample of enrolled cases. Genotyping of all 404 strains was done by Sanger's sequencing of the core region. The phylogenetic analysis of 179 HCV strains with a high-quality sequencing data was performed. RESULTS: The distributions of prevalent GTs/subtypes as noted in the current study were ( n [%]): GT1a, 101 (25%); GT1b, 12 (2.9%); GT1c, 1 (0.25%); GT3a, 275 (68.07%); GT3b, 9 (2.2%); GT3g, 2 (0.49%); GT3i, 3 (0.74%); and GT4a, 1 (0.24%). HCV GTs GT2, GT5, and GT6 were not detected from our region. Sequence analysis showed high genotypic variability in HCV GT3. Phylogenetic analysis showed that HCV GT3 and GT1 circulating in our region were related to Indian strains reported earlier. CONCLUSIONS: HCV GTs 3a and 1a are the commonest circulating GTs in Uttar Pradesh, India.


Assuntos
Variação Genética , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/virologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Técnicas de Genotipagem , Hepacivirus/isolamento & purificação , Antígenos do Núcleo do Vírus da Hepatite B/genética , Hepatite C/epidemiologia , Humanos , Índia/epidemiologia , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Filogenia , Prevalência , Estudos Retrospectivos , Análise de Sequência de DNA , Adulto Jovem
12.
Clin Chim Acta ; 486: 237-244, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30025756

RESUMO

BACKGROUND: Hepatitis B core-related antigen (HBcrAg) has been revealed as an important marker of Hepatitis B virus (HBV) infection recently. We aimed to evaluate the HBcrAg assay for indication of HBV loads in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) patients and assess the association between HBcrAg/cccDNA and HCC recurrence. METHODS: HBcrAg was measured by chemiluminescence enzyme immunoassay. Intrahepatic covalently closed circular DNA (cccDNA) was measured by real-time PCR with TaqMan fluorescent probes based on liver specimens from 89 HCC patients. RESULTS: HBcrAg correlated positively with HBV DNA irrespective of HBeAg status. Both HBcrAg and HBV DNA were associated with cccDNA in patients with elevated serum HBV DNA (>4 log IU/mL). In patients with non-elevated HBV DNA (≤4 log IU/mL), no relationship between HBV DNA and cccDNA was observed, but we still documented a modest correlation between HBcrAg and cccDNA. Finally, the recurrence-free survival rates were significantly lower in HCC patients with high intrahepatic cccDNA and serum HBcrAg levels than those with low cccDNA/HBcrAg levels (p = 0.035, p = 0.003 respectively). CONCLUSIONS: HBcrAg not only can serve as a biomarker to assess HBV loads in patients as well as provide a good method for monitoring cccDNA in HCC, but also can be used as a good prognostic predictor for HCC patients.


Assuntos
Carcinoma Hepatocelular/genética , DNA Viral/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Neoplasias Hepáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Estudos de Coortes , DNA Viral/sangue , Feminino , Corantes Fluorescentes/química , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Hepatite B Crônica/sangue , Hepatite B Crônica/diagnóstico , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Luminescência , Masculino , Pessoa de Meia-Idade , Adulto Jovem
13.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30045981

RESUMO

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Mutagênese , Pirofosfatases/genética , Ribavirina/farmacologia , Antivirais/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Nucleotídeos/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Ribavirina/metabolismo , Transdução de Sinais
14.
Cell Physiol Biochem ; 48(3): 1041-1059, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30041239

RESUMO

BACKGROUND/AIMS: Developing engineered dendritic cell (DC)-targeting lentivectors (LVs) have been the target of intense research for their potential to create antigen-directed immunotherapeutics which can be safely administered to patients. In this study, we constructed a DC-directed LV (LVDC-UbHBcAg-LIGHT) as a potential vaccine to induce anti-HBV immune responses. METHODS: Specificity of LVDC-UbHBcAg-LIGHT for DCs in vivo was confirmed through live animal imaging studies. The levels of cytokine production in T cells were assessed by flow cytometry. The HBcAg-specific cytotoxic T lymphocyte (CTL) responses and antibody responses induced by direct administration of the LVs were detected by LDH release assay and ELISA respectively. The levels of serum HBsAg and HBV DNA were evaluated by Abbott kits and quantitative PCR respectively. The expression levels of HBsAg and HBcAg in liver tissues of HBV transgenic mice were examined by immunohistochemistry. In addition, molecular mechanism underlying the activation of CD8+ T cells was explored. RESULTS: Live animal imaging studies showed that following subcutaneous administration of LVDC-UbHBcAg-LIGHT, no obvious luminescence signal was detected at the injection site. Immunization with LVDC-UbHBcAg-LIGHT elicited potent T cell responses in HBV transgenic mice evidenced by increased percentages of IFN-γ, TNF-α and GzmB producing CD8+ T cells as well as IFN-γ producing CD4+ T cells, improved HBcAg-specific CTL activities and antibody responses. Additionally, vaccination with LVDC-UbHBcAg-LIGHT efficiently reduced serum HBsAg, HBV DNA levels and the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice. More importantly, autophagy was induced in the activated CD8+ T cells, and the induced autophagy noticeably promoted the proliferation of T cells and decreased the frequencies of apoptotic CD8+ T cells by selectively degrading ubiquitinated apoptosis and cell cycle-associated protein aggregates. Futhermore, we confirmed the interaction between autophagosomes and ubiquitinated aggregates by confocal microscopy and immunoprecipitation analysis. CONCLUSIONS: These results demonstrated that LVDC-UbHBcAg-LIGHT provided a simple method of eliciting effective antiviral immune responses in HBV transgenic mice and might potentially be used as a therapeutic strategy to eradicate HBV with more safety and efficiency. Moreover, our results revealed a direct role of autophagy in promoting the survival and proliferation of activated CD8+ T cells.


Assuntos
Autofagia , Vetores Genéticos/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Linfócitos T Citotóxicos/metabolismo , Animais , Anticorpos Antivirais/sangue , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Vetores Genéticos/genética , Granzimas/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Humanos , Interferon gama/análise , Lentivirus/genética , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Replicação Viral
15.
Acta Virol ; 62(2): 157-163, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29895156

RESUMO

Hepatitis B virus (HBV) infection is a major public health problem and immune tolerance is responsible for persistent HBV infection. HBV therapeutic vaccines targeting HBV e antigen (HBeAg) may have an excellent effect in overcoming HBV immune tolerance. Thus, there is urgency for designing therapeutic vaccine candidates that target HBeAg. In this research, we fused the C (472-507) gene sequence of HBV with the extracellular domain of human CD40 ligand sequence and ligated this fused sequence into the pEGFP-N1 vector to construct the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L. Then, the dendritic cells (DCs) generated from human peripheral blood were transfected with this recombinant plasmid. After this, the phenotype and function of DCs were assessed. Compared with the three control groups of pEGFP-N1-C (472-507), pEGFP-N1 and phosphate buffered saline (PBS), we found that DCs transfected with the recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L enhanced the expression of costimulatory molecules (CD80, CD86 and HLA-DR) and secretion of cytokine IL-12p70. Furthermore, the capacity of inducing the proliferation of allogeneic lymphocytes was also improved. Our study validated that transfecting DCs with recombinant plasmid pEGFP-N1-C (472-507)-ecdCD40L could activate DCs and enhance their functions. Therefore, C (472-507)-ecdCD40L fusion sequence may be a promising vaccine candidate for chronic hepatitis B therapythat targets HBeAg.


Assuntos
Antígenos CD40/imunologia , Células Dendríticas/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Vacinas contra Hepatite Viral/imunologia , Antígenos CD40/química , Antígenos CD40/genética , Células Dendríticas/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatite B/prevenção & controle , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Domínios Proteicos , Transfecção , Vacinas contra Hepatite Viral/genética
16.
Methods Mol Biol ; 1776: 97-123, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29869237

RESUMO

The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98-114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent "spike." The tips of the "spikes" are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface "spike," thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins.Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Proteínas Recombinantes de Fusão/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Sequência de Aminoácidos , Bactérias/virologia , Epitopos/genética , Pichia/genética , Pichia/virologia , Plantas/virologia , Vacinas Virais/genética , Leveduras/virologia
17.
Methods Mol Biol ; 1776: 503-531, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29869263

RESUMO

The highly immunogenic icosahedral capsid of hepatitis B virus (HBV) can be exploited as a nanoparticulate display platform for heterologous molecules. Its constituent core protein (HBc) of only ~180 amino acids spontaneously forms capsid-like particles (CLPs) even in E. coli. The immunodominant c/e1 epitope in the center of the HBc primary sequence comprises a solvent-exposed loop that tolerates insertions of flexible peptide sequences yet also of selected whole proteins as long as their 3D structures fit into the two acceptor sites. This constraint is largely overcome in the SplitCore system, where the sequences flanking the loop are expressed as two separate but self-complementing entities, with the foreign sequence fixed to the carrier at one end only. Both the contiguous and the split type of CLP strongly enhance immunogenicity of the displayed sequence but also nonvaccine applications can easily be envisaged. After a brief survey of the basic features of the two HBc carrier forms, we provide conceptual guidelines concerning which foreign proteins are likely to be presentable, or not, on either carrier type. We describe generally applicable protocols for CLP expression in E. coli, cell lysis and CLP enrichment by sucrose gradient velocity sedimentation, plus a simple but meaningful gel electrophoretic assay to assess proper particle formation.


Assuntos
Proteínas do Capsídeo/química , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/química , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Nanopartículas/química
18.
Protein Expr Purif ; 151: 86-92, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29908914

RESUMO

Recombinant virus-like particles (VLPs) are proven to be safe and effective vaccine candidates. We have previously described a plant-based recombinant protein expression system based on agroinfiltration of a replicating vector derived from the geminivirus bean yellow dwarf virus (BeYDV). The system has been systematically optimized to improve expression and reduce cell death in Nicotiana benthamiana leaves. Using these modifications, we show that VLPs derived from genotype GII.4 norovirus, the leading cause of acute gastroenteritis worldwide, can be produced at >1 mg/g leaf fresh weight (LFW), over three times the highest level ever reported in plant-based systems. We also produced norovirus GI VLPs at 2.3 mg/g LFW. Treatment of VLP-containing crude leaf extracts with acid, detergent, or heat enhanced recovery and allowed selective enrichment of norovirus VLPs. Optimal treatment conditions allowed removal of >90% of endogenous plant proteins without any loss of norovirus VLPs. Selective enrichment of hepatitis B core antigen (HBcAg) VLPs by acid treatment was also demonstrated, with some losses in yield that were partially mitigated in the presence of detergent. Sedimentation analysis confirmed that acid and detergent did not inhibit proper assembly of norovirus VLPs, although heat treatment had a small negative effect. These results demonstrate that milligram quantities of norovirus VLPs can be obtained and highly enriched in a matter of days from a single plant leaf using the BeYDV plant expression system.


Assuntos
Geminiviridae/genética , Norovirus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Capsídeo/metabolismo , Vetores Genéticos , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/genética
19.
Arch Virol ; 163(10): 2829-2833, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29948380

RESUMO

The high prevalence (14.3%) of HIV/HBV co-infections reported in west China makes it necessary to screen concurrent HBV infection in HIV carriers. HBV B genotype was shown to be dominant in 54 cases of HIV/HBV co-infection, accounting for 81.48% of the total. The total drug resistance rate observed was 3.70%. A1762T, G1764A and G1896A mutations were common mutations identified in the BCP/PC region. However, the prevalence of the G1896A mutation was significantly high among the HBeAg negative HIV/HBV co-infected patients, and may be associated with high HBV replication. Mutations in the PC region are related to the loss in synthesis of HBeAg and may accelerate HBV replication in HIV positive patients.


Assuntos
Coinfecção/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Vírus da Hepatite B/genética , Hepatite B/virologia , Regiões Promotoras Genéticas , Replicação Viral , Adulto , Feminino , HIV-1/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação
20.
J Virol ; 92(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29950410

RESUMO

Under the immune pressure of cytotoxic T cells (CTLs), hepatitis B virus (HBV) evolves to accumulate mutations more likely within epitopes to evade immune detection. However, little is known about the specific patterns of the immune pressure-associated HBV mutation of T-cell epitopes and their link to disease progression. Here, we observed a correlation of the accumulated variants on HBV core protein (HBc) with the disease severity of HBV infection. Further analysis indicated that these substitutions were mostly located within CD8+ T-cell epitopes of HBc protein, which were systematically screened and identified in an unbiased manner in our study. From individual peptide level to the human leukocyte antigen I (HLA-I)-restricted population level, we elucidated that the mutations in these well-defined HLA-I-restricted T-cell epitopes significantly decreased antiviral activity-specific CTLs and were positively associated with clinical parameters and disease progression in HBV-infected patients. The molecular pattern for viral epitope variations based on the sequencing of 105 HBV virus genomes indicated that the C-terminal portion (Pc), especially the Pc-1 and Pc-2 positions, have the highest mutation rates. Further structural analysis of HLA-A*02 complexed to diverse CD8+ T-cell epitopes revealed that the highly variable C-terminal bulged peak of M-shaped HBc-derived epitopes are solvent exposed, and most of the CDR3ßs of the T-cell receptor hover over them. These data shed light on the molecular and immunological mechanisms of T-cell immunity-associated viral evolution in hepatitis B progression, which is beneficial for designing immunotherapies and vaccines.IMPORTANCE The specific patterns of sequence polymorphisms of T-cell epitopes and the immune mechanisms of the HBV epitope mutation-linked disease progression are largely unclear. In this study, we systematically evaluated the contribution of CD8+ T cells to the disease progress-associated evolution of HBV. By evaluation of patient T-cell responses based on the peptide repertoire, we comprehensively characterized the association of clinical parameters in chronic hepatitis B with the antiviral T-cell response-associated mutations of the viruses from the single-epitope level to the overall HLA-I-restricted peptide levels. Furthermore, we investigated the molecular basis of the HLA-A2-restricted peptide immune escape and found that the solvent-exposed C-terminal portion of the epitopes is highly variable under CDR3ß recognition. Our work may provide a comprehensive evaluation of viral mutations impacted by the host CTL response in HBV disease progression in the context of the full repertoire of HBc-derived epitopes.


Assuntos
Epitopos de Linfócito T/imunologia , Evolução Molecular , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Linfócitos T/imunologia , Epitopos de Linfócito T/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Mutação , Seleção Genética , Análise de Sequência de DNA
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